Hyaluronan receptor expressed in human umbilical vein endothelial

ABSTRACT

The present invention provides nucleotide and amino acid sequences that identify and encode the hyaluronan receptor (hr) from human umbilical vein endothelial cells. The present invention also provides for antisense molecules to the nucleotide sequences which encode hr, expression vectors for the production of purified HR, antibodies capable of binding specifically to HR, hybridization probes or oligonucleotides for detecting the upregulation of HR encoding nucleotide sequences, genetically engineered host cells for the expression of HR, diagnostic tests for activated, angiogenic, inflamed or metastatic cells and/or tissues based on HR-encoding nucleic acid molecules and antibodies capable of binding specifically to the receptor.

This application is a division of application Ser. No. 08/402,217, filedMar. 10, 1995, now U.S. Pat. No. 5,587,301.

OVERVIEW

All blood vessels are composed of three layers or tunics. The tunicaintima consists of endothelial cells which line the vessel and rest onthe basal lamina or middle layer. The subendothelial layer consists ofloose connective tissue and may contain smooth muscle cells. Theendothelial cells are generally polygonal and elongated in the directionof blood flow. The nucleus of the endothelial cell bulges into thecapillary lumen, and Golgi complex is located at the nuclear poles. Afew mitochondria, free ribosomes and rough endoplasmic reticulum arepresent. Endothelial cells are held together by zona occludentes and anoccasional desmosome; gap junctions which offer variable permeability tomacromolecules are present. The Weibel Palade body, a rod shapedcytoplasmic inclusion is characteristic of these cells.

Vascular endothelial cells play a central role in physiologicalhomeostasis, blood vessel permeability, and response to physiologic andpathologic stimuli. The endothelium is a primary target forcardiovascular risk factors such as high blood pressure, shear stress,and atherosclerosis. It is sensitive to endothelin, growth factors,interleukin 1, epinephrine, angiotensin, arginine vasopressin, heparin,bradykinin, acetylcholine, prostacyclin, etc.

Hyaluronan (HA) is a negatively charged, high molecular weight,connective tissue polysaccharide found in the extracellular matrix ofmost animal tissues. It is synthesized in the plasma membrane offibroblasts and other cells and catabolized locally as well as in thelymph nodes or liver sinusoids. HA is commonly isolated from thevitreous body of the eye, synovial fluid, umbilical cord, and skin. Ithas several physiological functions including roles in water and plasmaprotein homeostasis; mitosis, cell migration and differentiationincluding angiogenesis (Rooney P and Kumar S (1994) EXS (Switzerland)70:179-90); and tissue remodeling, either as a normal ortumor-associated event.

The matrix-induced effects on cells are directed by a wide variety ofHA-binding proteins, such as the hyaluronan receptor (HR). Thewidespread occurrence of HRs indicate their importance in tissueorganization and control of cellular behavior. The family is known asthe hyaladherins and includes those HA-binding proteins which act aspart of the structural matrix and those which interact with HA at theplasma membrane as cell-surface matrix receptors. With the recognitionof the hyaluronan cell-surface receptor (HR), cell biologists,pathologists, and immunologists have begun to investigate the importanceof the HA and HR for their potential diagnostic and therapeutic value.

Matrix Hyaladherins

HRs found within the cartilage matrix have been well characterized.Aggrecan is the large aggregating chondroitin sulfate proteoglycan ofcartilage which has a high affinity for HA. Link protein is a 45-48 kDaglycoprotein which also demonstrates strong specific binding affinity.HA may bind more than 100 aggrecan and link protein molecules in asupramolecular complex which confers the viscoelastic properties ofcartilage. Other matrix proteins such as PG-M and type VI collagen whichparticipate in assembly and integrity may also be involved.

HA-binding proteins are also found in noncartilaginous tissues. Versicanof fibroblasts, hyaluronectin of nervous and soft connective tissues,glial hyaluronan binding protein in the central nervous system, andneurocan, a chondroitin sulfate proteoglycan of brain also form strongstructural complexes with HA. All matrix hyaloadherins contain tandemrepeated B loops, a structural motif believed to contain the HA-bindingdomain.

HR hyaloadherins have been detected on several cell types from a widevariety of tissues. Some reports suggest that HR are related to the CD44family of lymphocyte homing receptors which include the isoforms, Pgp-1,Hermes antigen, H-CAM, ECMRIII, etc. The distal extracellular domain ofCD44 has sequence homology to one of the B loop motifs of link protein.The numerous isoforms suggest different cellular functions anddemonstrate binding to other ligands such as collagens I and IV andmucosal vascular addressin.

Other non-CD44 HR include cell-surface antigens termed IVd4 which blockbinding of HA, liver endothelial cell receptors (LEC) which are involvedin the clearance of HA from the circulation, and fibroblast-produced HRwhich may be located on the cell surface where it mediates HA-inducedcell locomotion. Its 58 kDA soluble form contains an HA-bindingcomponent unrelated to the B loop motif and is known as receptor for HAmediated motility (RHAMM). The important distinctions betweencell-surface and matrix hyaloadherins are 1) HA hexasaccharidesrepresent the minimum size molecule that interacts with thesecell-surface receptors, 2) binding affinity increases with increasingpolymer length, and 3) binding increases with increasing buffer ionicstrength.

Cell Migration

Increased matrix presence of HA has been correlated with cell migrationin embryogenesis, limb regeneration, wound healing and tumor invasion.Since the CD44 HR have been shown to associate with the cytoskeletalankyrin, proteins of the HR complex may affect reorganization of theactin cytoskeleton and other activities such as cell ruffling,detachment from the substratum, and locomotion necessary for cellmigration. RHAMM, as one of the HR complex proteins, binds to HA withhigh affinity and is expressed only in the leading lamellae andperinuclear regions of migrating fibroblasts. Since RHAMM does notinclude a transmembrane hydrophobic region, it is assumed to be aperipheral protein associated with intracellular, membrane-boundtyrosine kinase. In studies of timed administration of HA and aninhibitor of tyrosine kinase, HA stimulated locomotion via a rapidtyrosine kinase signal transduction pathway.

Tumor Invasion and Metastasis

Invasive or metastatic cancer cells have the capacity to exit from thevascular system by use of sets of molecules, at least one of whichalways has a receptor function. One series of such sets might includesuccessive interactions among endothelial VLA-4 integrin and E-selectin,subendothelial collagen IV and β-4 integrin, and soft connective tissueHA and CD44 or HR interactions (Akiyana et al (1993) Semin Cancer Biol4:215-218).

Some tumor cells also have the capacity to assemble HA-enrichedpericellular matrices which reduce cell adhesion to the outside of thegrowing tumor and protect the tumor from immune surveillance. Inaddition, the presence of high HA attracts endothelial cells which areactive in angiogenesis. The combination of these HA functions allows therapid establishment and growth of invasive tumor cells.

The transforming oncogene H-ras may promote cell locomotion. Hardwick etal (1992 J Cell Biol 117:1343-1350) reported that H-ras actuallyregulates expression of RHAMM, showed binding between HA and RHAMM, andproduced an antibody to the protein which is capable of inhibiting HA/HRlocomotion.

SUMMARY OF THE INVENTION

The subject invention provides nucleotide sequence which uniquelyencodes a novel human hyaluronan receptor. The cDNA, known as hr, wasfully contained within Incyte Clone No. 39200 and encodes a polypeptidedesignated HR.

The invention also comprises diagnostic tests for physiologic orpathologic compromise which include the steps of testing a sample or anextract with hr nucleic acids, fragments or oligomers thereof. Aspectsof the invention include the antisense DNA of hr; cloning or expressionvectors containing hr; host cells or organisms transformed withexpression vectors containing hr; a method for the production andrecovery of purified HR from host cells; and purified protein, HR, whichcan be used to generate antibodies and other molecules for diagnosis ofactivated, angiogenic, inflamed or metastatic cells and/or tissues.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid (aa) alignment of HR (SEQ ID NO:2) withmouse hyaluronan receptor (SEQ ID NO:3). The unmatched aa in the middleof the sequence may reflect the position of a mouse intron. The cDNAlacks the intron since it was constructed from mRNA. Alignments shownwere produced using the multisequence alignment program of DNASTARsoftware (DNASTAR Inc, Madison, WIS.).

FIG. 2 displays an analysis of HR hydrophobicity based on the predictedaa sequence and composition.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, HRs, refers to polypeptides, naturally occurring HRs andactive fragments thereof, which are encoded by mRNAs transcribed fromthe cDNA of Seq ID No 1.

"Active" refers to those forms of HR which retain the biologic and/orimmunologic activities of any naturally occurring HR.

"Naturally occurring HR" refers to HRs produced by human cells that havenot been genetically engineered and specifically contemplates variousHRs arising from post-translational modifications of the polypeptideincluding but not limited to acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation.

"Derivative" refers to HRs chemically modified by such techniques asubiquitination, labeling (e.g., with radionuclides, various enzymes,etc.), pegylation (derivatization with polyethylene glycol), andinsertion or substitution by chemical synthesis of aa such as ornithine,which do not normally occur in human proteins.

"Recombinant variant" refers to any polypeptide differing from naturallyoccurring HRs by aa insertions, deletions, and substitutions, createdusing recombinant DNA techniques. Guidance in determining which aaresidues may be replaced, added or deleted without abolishing activitiesof interest, such as cell adhesion and chemotaxis, may be found bycomparing the sequence of the particular HR with that of homologousreceptors and minimizing the number of aa sequence changes made inregions of high homology.

Preferably, aa "substitutions" are the result of replacing one aa withanother aa having similar structural and/or chemical properties, such asthe replacement of a leucine with an isoleucine or valine, an aspartatewith a glutamate, or a threonine with a serine, i.e., conservative aareplacements. "Insertions" or "deletions" are typically in the range ofabout 1 to 5 aa. The variation allowed may be experimentally determinedby systematically making insertions, deletions, or substitutions of aain a HR molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity.

Where desired, a "signal or leader sequence" can direct the polypeptidethrough the membrane of a cell. Such a sequence may be naturally presenton the polypeptides of the present invention or provided fromheterologous protein sources by recombinant DNA techniques.

A polypeptide "fragment," "portion," or "segment" is a stretch of aaresidues of at least about 5 amino acids, often at least about 7 aa,typically at least about 9 to 13 aa, and, in various embodiments, atleast about 17 or more aa. To be active, any HR polypeptide must havesufficient length to display biologic and/or immunologic activity.

An "oligonucleotide" or polynucleotide "fragment", "portion," or"segment" is a stretch of nucleotide residues which is long enough touse in polymerase chain reaction (PCR) or various hybridizationprocedures to amplify or simply reveal related parts of mRNA or DNAmolecules.

The present invention includes purified HR polypeptide from natural orrecombinant sources, cells transformed with recombinant nucleic acidmolecules encoding HR. Various methods for the isolation of HRpolypeptide may be accomplished by procedures well known in the art. Forexample, such polypeptide may be purified by immunoaffinitychromatography by employing the antibodies provided by the presentinvention. Various other methods of protein purification well known inthe art include those described in Deutscher M (1990) Methods inEnzymology, Vol 182, Academic Press, San Diego; and Scopes R (1982)Protein Purification: Principles and Practice, Springer-Verlag, NYC,both incorporated herein by reference.

"Recombinant" may also refer to a polynucleotide which encodes HR and isprepared using recombinant DNA techniques. The DNA which encodes HR mayalso include allelic or recombinant variants and mutants thereof.

"Oligonucleotides" or "nucleic acid probes" are prepared based on thecDNA sequence which encodes HR provided by the present invention.Oligonucleotides comprise portions of the DNA sequence having at leastabout 15 nucleotides, usually at least about 20 nucleotides. Nucleicacid probes comprise portions of the sequence having fewer nucleotidesthan about 6 kb, usually fewer than about 1 kb. After appropriatetesting to eliminate false positives, these probes may be used todetermine whether mRNAs encoding HR are present in a cell or tissue orto isolate similar nucleic acid sequences from chromosomal DNA asdescribed by Walsh PS et al (1992 PCR Methods Appl 1:241-250).

Probes may be derived from naturally occurring or recombinant single- ordouble-stranded nucleic acids or be chemically synthesized. They may belabeled by nick translation, Klenow fill-in reaction, PCR or othermethods well known in the art. Probes of the present invention, theirpreparation and/or labeling are elaborated in Sambrook J et al (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,N.Y.; or Ausubel FM et al (1989) Current Protocols in Molecular Biology,John Wiley & Sons, NYC, both incorporated herein by reference.

"Activated" cells as used in this application are those which areengaged in migration, proliferation, vascularization or differentiationas part of a normal or disease process.

Recombinant variants encoding these same or similar polypeptides may besynthesized or selected by making use of the "redundancy" in the geneticcode. Various codon substitutions, such as the silent changes whichproduce various restriction sites, may be introduced to optimize cloninginto a plasmid or viral vector or expression in a particular prokaryoticor eukaryotic system. Mutations may also be introduced to modify theproperties of the polypeptide, to change ligand-binding affinities,interchain affinities, or polypeptide degradation or turnover rate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a nucleotide sequence uniquelyidentifying a novel human hyaluronan receptor, HR, which was highlyexpressed in the human umbilical vein endothelial library. Because HR isspecifically expressed in embryonic tissue, the nucleic acid (hr),polypeptide (HR) and antibodies to HR are useful in diagnostic assaysfor invasive cancers. Excessive expression of HR can direct cellmigration, including lymphocytes and/or other cells which respond tohyaluronan. Therefore, a diagnostic test for excess expression of HR canaccelerate diagnosis and proper treatment of an abnormal conditioncaused by viral or other infections; angiogenesis of cancerous tissues;invasive leukemias and lymphomas; or other physiologic/pathologicproblems which deviate from normal development and result in metastaticcell migration, proliferation, vascularization and differentiation.

The nucleotide sequence encoding HR (or its complement) has numerousapplications in techniques known to those skilled in the art ofmolecular biology. These techniques include use as hybridization probes,use as oligomers for PCR, use for chromosome and gene mapping, use inthe recombinant production of HR, and use in generation of anti-senseDNA or RNA, their chemical analogs and the like. Uses of the nucleotidesequences encoding HR disclosed herein are exemplary of known techniquesand are not intended to limit their use in any technique known to aperson of ordinary skill in the art. Furthermore, the nucleotidesequences disclosed herein may be used in molecular biology techniquesthat have not yet been developed, provided the new techniques rely onproperties of nucleotide sequences that are currently known, e.g., thetriplet genetic code, specific base pair interactions, etc.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of HR-encodingnucleotide sequences, some bearing minimal homology to the nucleotidesequence of any known and naturally occurring gene may be produced. Theinvention has specifically contemplated each and every possiblevariation of nucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe nucleotide sequence of naturally occurring HR, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HR and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HR gene under stringent conditions, it may beadvantageous to produce nucleotide sequences encoding HR or itsderivatives possessing a substantially different codon usage. Codons canbe selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic expression host inaccordance with the frequency with which particular codons are utilizedby the host. Other reasons for substantially altering the nucleotidesequence encoding HR and its derivatives without altering the encoded aasequence include the production of RNA transcripts having more desirableproperties, such as a greater half-life, than transcripts produced fromthe naturally occurring sequence.

The nucleotide sequence encoding HR may be joined to a variety of othernucleotide sequences by means of well established recombinant DNAtechniques (cf Sambrook J et al. (1989) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y.). Useful nucleotidesequences for joining to hr include an assortment of cloning vectors,e.g., plasmids, cosmids, lambda phage derivatives, phagemids, and thelike, that are well known in the art. Vectors of interest includeexpression vectors, replication vectors, probe generation vectors,sequencing vectors, and the like. In general, vectors of interest maycontain an origin of replication functional in at least one organism,convenient restriction endonuclease sensitive sites, and selectablemarkers for the host cell.

Another aspect of the subject invention is to provide for hr-specificnucleic acid hybridization probes capable of hybridizing with naturallyoccurring nucleotide sequences encoding HR. Such probes may also be usedfor the detection of similar hyaluronan receptor encoding sequences andshould preferably contain at least 50% of the nucleotides from this hrencoding sequence. The hybridization probes of the subject invention maybe derived from the nucleotide sequence of the SEQ ID NO 1 or fromgenomic sequence including promoter, enhancer elements and introns ofthe respective naturally occurring hrs. Hybridization probes may belabeled by a variety of reporter groups, including radionuclides such as32P or 35S, or enzymatic labels such as alkaline phosphatase coupled tothe probe via avidin/biotin coupling systems, and the like.

PCR as described in U.S. Pat. Nos. 4,683,195; 4,800,195; and 4,965,188provides additional uses for oligonucleotides based upon the nucleotidesequences which encode HR. Such probes used in PCR may be of recombinantorigin, may be chemically synthesized, or may be a mixture of both andcomprise a discrete nucleotide sequence for diagnostic use or adegenerate pool of possible sequences for identification of closelyrelated genomic sequences.

Other means of producing specific hybridization probes for hr DNAsinclude the cloning of nucleic acid sequences encoding HR or HRderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art and are commercially available and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerase as T7 or SP6 RNA polymerase and theappropriate radioactively labeled nucleotides.

It is now possible to produce a DNA sequence, or portions thereof,encoding HR and its derivatives entirely by synthetic chemistry, afterwhich the gene can be inserted into any of the many available DNAvectors using reagents, vectors and cells that are known in the art atthe time of the filing of this application. Moreover, syntheticchemistry may be used to introduce mutations into the hr sequences orany portion thereof.

The nucleotide sequence can be used to construct an assay to detectinflammation or disease associated with abnormal levels of expression ofHR. The nucleotide sequence can be labeled by methods known in the artand added to a fluid or tissue sample from a patient under hybridizingconditions. After an incubation period, the sample is washed with acompatible fluid which optionally contains a dye (or other labelrequiring a developer) if the nucleotide has been labeled with anenzyme. After the compatible fluid is rinsed off, the dye is quantitatedand compared with a standard. If the amount of dye is significantlyelevated, the nucleotide sequence has hybridized with the sample, andthe assay indicates the presence of inflammation and/or disease.

The nucleotide sequence for hr can be used to construct hybridizationprobes for mapping that gene. The nucleotide sequence provided hereinmay be mapped to a chromosome and specific regions of a chromosome usingwell known genetic and/or chromosomal mapping techniques. Thesetechniques include in situ hybridization, linkage analysis against knownchromosomal markers, hybridization screening with libraries orflow-sorted chromosomal preparations specific to known chromosomes, andthe like. The technique of fluorescent in situ hybridization ofchromosome spreads has been described, among other places, in Verma etal (1988) Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York, N.Y.

Fluorescent in situ hybridization of chromosomal preparations and otherphysical chromosome mapping techniques may be correlated with additionalgenetic map data. Examples of genetic map data can be found in the 1994Genome Issue of Science (265:1981f). Correlation between the location ofhr on a physical chromosomal map and a specific disease (orpredisposition to a specific disease) can help delimit the region of DNAassociated with that genetic disease. The nucleotide sequence of thesubject invention may be used to detect differences in gene sequencebetween normal and carrier or affected individuals.

The nucleotide sequence encoding HR may be used to produce purified HRusing well known methods of recombinant DNA technology. Among the manypublications that teach methods for the expression of genes after theyhave been isolated is Goeddel (1990) Gene Expression Technology, Methodsand Enzymology, Vol 185, Academic Press, San Diego. HR may be expressedin a variety of host cells, either prokaryotic or eukaryotic. Host cellsmay be from the same species in which hr nucleotide sequences areendogenous or from a different species. Advantages of producing HR byrecombinant DNA technology include obtaining adequate amounts of theprotein for purification and the availability of simplified purificationprocedures.

Cells transformed with DNA encoding HR may be cultured under conditionssuitable for the expression of hyaluronan receptors and recovery of theprotein from the cell culture. HR produced by a recombinant cell may besecreted or may be contained intracellularly, depending on theparticular genetic construction used. In general, it is more convenientto prepare recombinant proteins in secreted form. Purification stepsvary with the production process and the particular protein produced.

In addition to recombinant production, fragments of HR may be producedby direct peptide synthesis using solid-phase techniques (cf Stewart etal (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco;Merrifield J (1963) J Am Chem Soc 85:2149-2154). In vitro proteinsynthesis may be performed using manual techniques or by automation.Automated synthesis may be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Foster City, Calif.) in accordancewith the instructions provided by the manufacturer. Various fragments ofHR may be chemically synthesized separately and combined using chemicalmethods to produce the full length molecule.

HR for antibody induction does not require biological activity; however,the protein must be immunogenic. Peptides used to induce specificantibodies may have an aa sequence consisting of at least five aa,preferably at least 10 aa. They should mimic a portion of the aasequence of the protein and may contain the entire aa sequence of asmall naturally occurring molecules like HR. Short stretches of HR aamay be fused with those of another protein such as keyhole limpethemocyanin and antibody produced against the chimeric molecule.

Antibodies specific for HR may be produced by inoculation of anappropriate animal with the polypeptide or an antigenic fragment. Anantibody is specific for HR if it is produced against an epitope of thepolypeptide and binds to at least part of the natural or recombinantprotein. Antibody production includes not only the stimulation of animmune response by injection into animals, but also analogous steps inthe production of synthetic antibodies or other specific-bindingmolecules such as the screening of recombinant immunoglobulin libraries(cf Orlandi R et al (1989) PNAS 86:3833-3837, or Huse WD et al (1989)Science 256:1275-1281) or the in vitro stimulation of lymphocytepopulations. Current technology (Winter G and Milstein C (1991) Nature349:293-299) provides for a number of highly specific binding reagentsbased on the principles of antibody formation. These techniques may beadapted to produce molecules specifically binding HR.

An additional embodiment of the subject invention is the use of HRspecific antibodies, inhibitors, or their analogs as bioactive agents totreat viral or other infections; angiogenesis of cancerous tissues;invasive leukemias and lymphomas; or other physiologic/pathologicproblems which deviate from normal development and result in metastaticcell migration, proliferation, vascularization and differentiation.

Bioactive compositions comprising agonists, antagonists, or inhibitorsof HR may be administered in a suitable therapeutic dose determined byany of several methodologies including clinical studies on mammalianspecies to determine maximum tolerable dose and on normal human subjectsto determine safe dosage. Additionally, the bioactive agent may becomplexed with a variety of well established compounds or compositionswhich enhance stability or pharmacological properties such as half-life.It is contemplated that the therapeutic, bioactive composition may bedelivered by intravenous infusion into the bloodstream or any othereffective means which could be used for treating invasive cancers.

The examples below are provided to illustrate the subject invention.These examples are provided by way of illustration and are not includedfor the purpose of limiting the invention.

EXAMPLES

I Isolation of mRNA and Construction of the cDNA Library

The hyaluronan receptor cDNA sequence was identified among the sequencescomprising the HUVEC library. The HUVEC cell line is a normal,homogeneous, well-characterized, early passage, endothelial cell culturefrom human umbilical vein (Cell Systems Corporation, 12815 NE 124th St,Kirkland, Wash. 98034).

The HUVEC CDNA library was custom constructed by Stratagene (11099 M.Torrey Pines Rd., La Jolla, Calif. 92037). cDNA synthesis was primedwith oligo dT hexamers, and synthetic adaptor oligonucleotides wereligated onto the cDNA ends to enable its insertion into Uni-ZAP™ vectorsystem (Stratagene). This allowed high efficiency unidirectional (senseorientation) lambda library construction and the convenience of aplasmid system with blue/white color selection to detect clones withcDNA insertions.

The quality of the cDNA library was screened using DNA probes, and then,the pBluescript® phagemid (Stratagene) was excised. This phagemid allowsthe use of a plasmid system for easy insert characterization,sequencing, site-directed mutagenesis, the creation of unidirectionaldeletions and expression of fusion polypeptides. Subsequently, thecustom-constructed library phage particles were infected into E. colihost strain XL1-Blue® (Stratagene). The high transformation efficiencyof this bacterial strain increases the probability that the cDNA librarywill contain rare, under-represented clones. Alternative unidirectionalvectors might include, but are not limited to, pcDNAI (Invitrogen) andpSHlox-1 (Novagen).

II Isolation of cDNA Clones

The phagemid forms of individual cDNA clones were obtained by the invivo excision process, in which XL1-BLUE was coinfected with an f1helper phage. Proteins derived from both lambda phage and f1 helperphage initiated new DNA synthesis from defined sequences on the lambdatarget DNA and create a smaller, single-stranded circular phagemid DNAmolecule that includes all DNA sequences of the pBluescript plasmid andthe cDNA insert. The phagemid DNA was released from the cells andpurified, then used to reinfect fresh bacterial host cells (SOLR,Stratagene), where the double-stranded phagemid DNA was produced.Because the phagemid carries the gene for β-lactamase, the newlytransformed bacteria were selected on medium containing ampicillin.

Phagemid DNA was purified using the QIAWELL-8 Plasmid PurificationSystem from QIAGEN® DNA Purification System (QIAGEN Inc, 9259 Eton Ave,Chatsworth, Calif. 91311). This technique provides a rapid and reliablehigh-throughput method for lysing the bacterial cells and isolatinghighly purified phagemid DNA. The DNA eluted from the purification resinwas suitable for DNA sequencing and other analytical manipulations.

III Sequencing of cDNA Clones

The CDNA inserts from random isolates of the HUVEC library weresequenced in part. Methods for DNA sequencing are well known in the art.Conventional enzymatic methods employed DNA polymerase Klenow fragment,SEQUENASE® (US Biochemical Corp, Cleveland, Ohio) or Taq polymerase toextend DNA chains from an oligonucleotide primer annealed to the DNAtemplate of interest. Methods have been developed for the use of bothsingle- and double-stranded templates. The chain termination reactionproducts were electrophoresed on urea-acrylamide gels and detectedeither by autoradiography (for radionuclide-labeled precursors) or byfluorescence (for fluorescent-labeled precursors). Recent improvementsin mechanized reaction preparation, sequencing and analysis using thefluorescent detection method have permitted expansion in the number ofsequences that can be determined per day (using machines such as theCatalyst 800 and the Applied Biosystems 373 DNA sequencer).

IV Homology Searching of cDNA Clones and Deduced Proteins

Each sequence so obtained was compared to sequences in GenBank using asearch algorithm developed by Applied Biosystems Inc. and incorporatedinto the INHERIT™ 670 Sequence Analysis System. In this algorithm,Pattern Specification Language (developed by TRW Inc.) was used todetermine regions of homology. The three parameters that determine howthe sequence comparisons run were window size, window offset, and errortolerance. Using a combination of these three parameters, the DNAdatabase was searched for sequences containing regions of homology tothe query sequence, and the appropriate sequences were scored with aninitial value. Subsequently, these homologous regions were examinedusing dot matrix homology plots to distinguish regions of homology fromchance matches. Smith-Waterman alignments were used to display theresults of the homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT™ 670 Sequence Analysis System in a way similar to that used inDNA sequence homologies. Pattern Specification Language and parameterwindows were used to search protein databases for sequences containingregions of homology which were scored with an initial value. Dot-matrixhomology plots were examined to distinguish regions of significanthomology from chance matches

V Identification, Full Length Sequencing and Translation of the Gene

INHERIT™ analysis of the randomly picked and sequenced portions ofclones from the HUVEC library identified the partial sequence fromIncyte 39200 as homologous to hyaluronan receptor from mouse (Hardwicket al (1992) J Cell Biol 117:1343 1350). The cDNA insert comprisingIncyte 39200 was fully sequenced using the same methods described above.The coding region of the insert (ATG->TGA) was identified and is shownas Sequence ID No. 1. This sequence for human hr was translated usingDNASTAR software, the in-frame translation was identified, and is shownin Sequence ID No. 2. When all three possible predicted translations ofthe sequence were searched against protein databases such as SwissProtand PIR, no exact matches were found to the possible translations of hr.FIG. 1 shows the degree of amino acid homology between HR and mouseRHAMM. The unmatched aa in the middle of the sequence may reflect theposition of a mouse intron. The cDNA lacks the intron since it wasconstructed from mRNA. FIG. 2 shows the hydrophobicity plot for HR.

VI Antisense Analysis

Knowledge of the correct, complete cDNA sequence of HR will enable itsuse in antisense technology in the investigation of gene function.Either oligonucleotides, genomic or cDNA fragments comprising theantisense strand of hr can be used either in vitro or in vivo to inhibitexpression of the mRNA. Such technology is now well known in the art,and probes can be designed at various locations along the nucleotidesequences. By treatment of cells or whole test animals with suchantisense sequences, the gene of interest can be effectively turned off.Frequently, the function of the gene can be ascertained by observingbehavior at the cellular, tissue or organismal level (e.g. lethality,loss of differentiated function, changes in morphology, etc.).

In addition to using sequences constructed to interrupt transcription ofthe open reading frame, modifications of gene expression can be obtainedby designing antisense sequences to intron regions, promoter/enhancerelements, or even to trans-acting regulatory genes. Similarly,inhibition can be achieved using Hogeboom base-pairing methodology, alsoknown as "triple helix" base pairing.

VII Expression of HR

Expression of hr may be accomplished by subcloning the cDNAs intoappropriate expression vectors and transfecting the vectors into anappropriate expression hosts. In this particular case, the cloningvector previously used for the generation of the tissue library alsoprovides for direct expression of hr sequences in E. coli. Upstream ofthe cloning site, this vector contains a promoter for β-galactosidase,followed by sequence containing the amino-terminal Met and thesubsequent 7 residues of β-galactosidase. Immediately following theseeight residues is an engineered bacteriophage promoter useful forartificial priming and transcription and a number of unique restrictionsites, including Eco RI, for cloning.

Induction of the isolated, transfected bacterial strain with IPTG usingstandard methods will produce a fusion protein corresponding to thefirst seven residues of β-galactosidase, about 15 residues of "linker",and the peptide encoded within the cDNA. Since cDNA clone inserts aregenerated by an essentially random process, there is one chance in threethat the included cDNA will lie in the correct frame for propertranslation. If the cDNA is not in the proper reading frame, it can beobtained by deletion or insertion of the appropriate number of bases bywell known methods including in vitro mutagenesis, digestion withexonuclease III or mung bean nuclease, or oligonucleotide linkerinclusion.

The hr cDNA can be shuttled into other vectors known to be useful forexpression of protein in specific hosts. Oligonucleotide amplimerscontaining cloning sites as well as a segment of DNA sufficient tohybridize to stretches at both ends of the target cDNA (25 bases) can besynthesized chemically by standard methods. These primers can then beused to amplify the desired gene segments by PCR. The resulting new genesegments can be digested with appropriate restriction enzymes understandard conditions and isolated by gel electrophoresis. Alternately,similar gene segments can be produced by digestion of the cDNA withappropriate restriction enzymes and filling in the missing gene segmentswith chemically synthesized oligonucleotides. Segments of the codingsequence from more than one gene can be ligated together and cloned inappropriate vectors to optimize expression of recombinant sequence.

Suitable expression hosts for such chimeric molecules include but arenot limited to mammalian cells such as Chinese Hamster Ovary (CHO) andhuman 293 cells, insect cells such as Sf9 cells, yeast cells such asSaccharomyces cerevisiae, and bacteria such as E. coli. For each ofthese cell systems, a useful expression vector may also include anorigin of replication to allow propagation in bacteria and a selectablemarker such as the β-lactamase antibiotic resistance gene to allowselection in bacteria. In addition, the vectors may include a secondselectable marker such as the neomycin phosphotransferase gene to allowselection in transfected eukaryotic host cells. Vectors for use ineukaryotic expression hosts may require RNA processing elements such as3' polyadenylation sequences if such are not part of the cDNA ofinterest.

Additionally, the vector may contain promoters or enhancers whichincrease gene expression. Such promoters are host specific and includeMMTV, SV40, or metallothionine promoters for CHO cells; trp, lac, tac orT7 promoters for bacterial hosts, or alpha factor, alcohol oxidase orPGH promoters for yeast. Transcription enhancers, such as the roussarcoma virus (RSV) enhancer, may be used in mammalian host cells. Oncehomogeneous cultures of recombinant cells are obtained through standardculture methods, large quantities of recombinantly produced HR can berecovered from the conditioned medium and analyzed using chromatographicmethods known in the art.

VIII Isolation of Recombinant HR

HR may be expressed as a chimeric protein with one or more additionalpolypeptide domains added to facilitate protein purification. Suchpurification facilitating domains include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, protein A domains that allowpurification on immobilized immunoglobulin, and the domain utilized inthe FLAGS extension/affinity purification system (Immunex Corp.,Seattle, Wash.). The inclusion of a cleavable linker sequence such asFactor XA or enterokinase(Invitrogen, San Diego, Calif.) between thepurification domain and the hr sequence may be useful to facilitateexpression of HR.

IX Production of HR Specific Antibodies

Two approaches are utilized to raise antibodies to HR, and each approachis useful for generating either polyclonal or monoclonal antibodies. Inone approach, denatured protein from the reverse phase HPLC separationis obtained in quantities up to 75 mg. This denatured protein can beused to immunize mice or rabbits using standard protocols; about 100micrograms are adequate for immunization of a mouse, while up to 1 mgmight be used to immunize a rabbit. For identifying mouse hybridomas,the denatured protein can be radioiodinated and used to screen potentialmurine B-cell hybridomas for those which produce antibody. Thisprocedure requires only small quantities of protein, such that 20 mgwould be sufficient for labeling and screening of several thousandclones.

In the second approach, the amino acid sequence of HR, as deduced fromtranslation of the cDNA, is analyzed to determine regions of highimmunogenicity. Oligopeptides comprising appropriate hydrophilicregions, as illustrated in FIG. 2, are synthesized and used in suitableimmunization protocols to raise antibodies. Analysis to selectappropriate epitopes is described by Ausubel FM et al (1989, CurrentProtocols in Molecular Biology, John Wiley & Sons, New York, N.Y.). Theoptimal amino acid sequences for immunization are usually at theC-terminus, the N-terminus and those intervening, hydrophilic regions ofthe polypeptide which are likely to be exposed to the externalenvironment when the protein is in its natural conformation.

Typically, selected peptides, about 15 residues in length, aresynthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH,Sigma) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester(MBS; cf. Ausubel FM et al, supra). If necessary, a cysteine may beintroduced at the N-terminus of the peptide to permit coupling to KLH.Rabbits are immunized with the peptide-KLH complex in complete Freund'sadjuvant. The resulting antisera are tested for antipeptide activity bybinding the peptide to plastic, blocking with 1% BSA, reacting withantisera, washing and reacting with labeled (radioactive orfluorescent), affinity purified, specific goat anti-rabbit IgG.

Hybridomas may also be prepared and screened using standard techniques.Hybridomas of interest are detected by screening with labeled HR toidentify those fusions producing the monoclonal antibody with thedesired specificity. In a typical protocol, wells of plates (FAST;Becton-Dickinson, Palo Alto, Calif.) are coated with affinity purified,specific rabbit-anti-mouse (or suitable anti-species Ig) antibodies at10 mg/ml. The coated wells are blocked with 1% BSA, washed and exposedto supernatants from hybridomas. After incubation the wells are exposedto labeled HR at 1 mg/ml. Clones producing antibodies will bind aquantity of labeled HR which is detectable above background. Such clonesare expanded and subjected to 2 cycles of cloning at limiting dilution(1 cell/3 wells). Cloned hybridomas are injected into pristine mice toproduce ascites, and monoclonal antibody is purified from mouse asciticfluid by affinity chromatography on Protein A. Monoclonal antibodieswith affinities of at least 10e8 Me-1, preferably 10e9 to 10e10 orstronger, will typically be made by standard procedures as described inHarlow and Lane (1988) Antibodies: A Laboratory Manual. Cold SpringHarbor Laboratory NY; and in Goding (1986) Monoclonal Antibodies:Principles and Practice, Academic Press, New York, N.Y., bothincorporated herein by reference.

Diagnostic Test Using HR Specific Antibodies

Particular HR antibodies are useful for the diagnosis of prepathologicconditions, and chronic or acute diseases which are characterized bydifferences in the amount or distribution of HR, respectively. To date,HR has only been found in the HUVEC library and is thus specific forabnormalities or pathologies which affect embryonic, angiogenic orinvasive cells.

Diagnostic tests for HR include methods utilizing the antibody and alabel to detect HR in human body fluids, tissues or extracts of suchtissues. The polypeptides and antibodies of the present invention may beused with or without modification. Frequently, the polypeptides andantibodies will be labeled by joining them, either covalently ornoncovalently, with a substance which provides for a detectable signal.A wide variety of labels and conjugation techniques are known and havebeen reported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent agents, chemiluminescent agents, magneticparticles and the like. Patents teaching the use of such labels includeU.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;4,275,149; and 4,366,241. Also, recombinant immunoglobulins may beproduced as shown in U.S. Pat. No. 4,816,567, incorporated herein byreference.

A variety of protocols for measuring soluble or membrane-bound HR, usingeither polyclonal or monoclonal antibodies specific for the protein, areknown in the art. Examples include enzyme-linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting(FACS). A two-site monoclonal-based immunoassay utilizing monoclonalantibodies reactive to two non-interfering epitopes on HR is preferred,but a competitive binding assay may be employed. These assays aredescribed, among other places, in Maddox, DE et al (1983, J Exp Med158:1211).

XI Purification of Native HR Using Specific Antibodies

Native or recombinant HR can be purified by immunoaffinitychromatography using antibodies specific for HR. In general, animmunoaffinity column is constructed by covalently coupling the anti-HRantibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either byprecipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated Sepharose (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

Such immunoaffinity columns were utilized in the purification of HR bypreparing a fraction from cells containing HR in a soluble form. Thispreparation was derived by solubilization of the whole cell or of asubcellular fraction obtained via differential centrifugation by theaddition of detergent or by other methods well known in the art.Alternatively, soluble HR containing a signal sequence may be secretedin useful quantity into the medium in which the cells are grown.

A soluble HR-containing preparation was passed over the immunoaffinitycolumn, and the column was washed under conditions that allow thepreferential absorbance of HR (eg, high ionic strength buffers in thepresence of detergent). Then, the column was eluted under conditionsthat disrupt antibody/HR binding (e.g., a buffer of pH 2-3 or a highconcentration of a chaotrope such as urea or thiocyanate ion), and HRwas collected.

XII Hyaluronan Induced Chemotaxis for Cell Activation and Wound Healing

The chemotactic interactions between HA and HR were measured in a48-well microchemotaxis chambers (cf. Falk WR et al (1980) J ImmunolMethods 33:239). In each well, two compartments are separated by afilter that allows the passage of cells in response to a chemicalgradient. Cells expressing HR in a culture medium such as RPMI 1640(Sigma, St. Louis, Mo.) are placed on one side of a filter, usuallypolycarbonate, and cells producing HA or a solution enriched with HA areplaced on the opposite side of the filter. Sufficient incubation time isallowed for the cells to traverse the filter in response to theconcentration gradient across the filter. Filters are recovered fromeach well, and cells adhering to the side of the filter facing the HAare typed and quantified.

Those cells producing HR and migrating toward the higher end of the HAgradient are rated for chemotactic specificity. This assay not onlysubstantiates the ability of HR-producing cells to respond to HA, butprovides researchers using methods well known in the art with modelsystems from which to obtain and describe transcription factors andenhancers specific for use in regulating HR activity in native cellpopulations. The ability to artificially supply such factors dissolvedin dimethyl sulfoxide (DMSO) or some other carrier liquid, to upregulateproduction of HR in a localized manner, and to enhance migrationcapability provides for the use of HA as a stimulant in wound healing.HA could be incorporated in collagenous or other natural or artificialbandage materials used to treat refractory wounds. The presence of HAwould attract activated endothelial cells, fibroblasts, etc. which wouldparticipate in the repair and healing process.

XIII Drug Screening

This invention is particularly useful for screening compounds by usingHR or binding fragments thereof in any of a variety of drug screeningtechniques. The polypeptide or fragment employed in such a test mayeither be free in solution, affixed to a solid support, borne on a cellsurface or located intracellularly. One method of drug screeningutilizes eukaryotic or prokaryotic host cells which are stablytransformed with recombinant nucleic acids expressing the polypeptide orfragment. Drugs are screened against such transformed cells incompetitive binding assays. Such cells, either in viable or fixed form,can be used for standard binding assays. One may measure, for example,the formation of complexes between HR and the agent being tested.Alternatively, one can examine the diminution in complex formationbetween HR and hyaluronan caused by the agent being tested.

Thus, the present invention provides methods of screening for drugs orany other agents which can affect cell migration, angiogenesis orinfiltration of lymphomas or leukemias. These methods comprisecontacting such an agent with HR polypeptide or a fragment thereof andassaying (i) for the presence of a complex between the agent and the HRpolypeptide or fragment, or (ii) for the presence of a complex betweenthe HR polypeptide or fragment and the cell, by methods well known inthe art. In such competitive binding assays, the HR polypeptide orfragment is typically labeled. After suitable incubation, free HRpolypeptide or fragment is separated from that present in bound form,and the amount of free or uncomplexed label is a measure of the abilityof the particular agent to bind to HR or to interfere with the HR andagent complex.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the HR polypeptidesand is described in detail in European Patent Application 84/03564,published on Sep. 13, 1984, incorporated herein by reference. Brieflystated, large numbers of different small peptide test compounds aresynthesized on a solid substrate, such as plastic pins or some othersurface. The peptide test compounds are reacted with HR polypeptide andwashed. Bound HR polypeptide is then detected by methods well known inthe art. Purified HR can also be coated directly onto plates for use inthe aforementioned drug screening techniques. In addition,non-neutralizing antibodies can be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding HRspecifically compete with a test compound for binding to HR polypeptidesor fragments thereof. In this manner, the antibodies can be used todetect the presence of any peptide which shares one or more antigenicdeterminants with HR.

XIV Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides of interest or of small molecules withwhich they interact, e.g., agonists, antagonists, or inhibitors. Any ofthese examples can be used to fashion drugs which are more active orstable forms of the polypeptide or which enhance or interfere with thefunction of a polypeptide in vivo (cf Hodgson J (1991) Bio/Technology9:19-21, incorporated herein by reference).

In one approach, the three-dimensional structure of a protein ofinterest, or of a protein-inhibitor complex, is determined by x-raycrystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of thepolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of a polypeptide may be gained by modeling basedon the structure of homologous proteins. In both cases, relevantstructural information is used to design efficient inhibitors. Usefulexamples of rational drug design may include molecules which haveimproved activity or stability as shown by Braxton S and Wells JA (1992Biochemistry 31:7796-7801) or which act as inhibitors, agonists, orantagonists of native peptides as shown by Athauda SB et al (1993 JBiochem 113:742-746), incorporated herein by reference.

It is also possible to isolate a target-specific antibody, selected byfunctional assay, as described above, and then to solve its crystalstructure. This approach, in principle, yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies(anti-ids) to a functional, pharmacologically active antibody. As amirror image of a mirror image, the binding site of the anti-ids wouldbe expected to be an analog of the original receptor. The anti-id couldthen be used to identify and isolate peptides from banks of chemicallyor biologically produced peptides. The isolated peptides would then actas the pharmacore.

By virtue of the present invention, sufficient amount of polypeptide maybe made available to perform such analytical studies as X-raycrystallography. In addition, knowledge of the HR amino acid sequenceprovided herein will provide guidance to those employing computermodeling techniques in place of or in addition to x-ray crystallography.

XV Identification of Other Members of the HR Complex

Purified HR is useful for characterization and purification ofassociated cell surface receptors and binding molecules. Cells whichrespond to HA by chemotaxis or other specific responses are likely toexpress a receptor for HR and to interact with transmembrane signalingmolecules such as tyrosine kinase. Radioactive labels may beincorporated into HR by various methods known in the art. A preferredembodiment is the labeling of primary amino groups in HR with ¹²⁵Bolton-Hunter reagent (Bolton, AE and Hunter, WM (1973) Biochem J 133:529), which has been used to label other signaling molecules withoutconcomitant loss of biological activity (Hebert CA et al (1991) J BiolChem 266: 18989; McColl S et al (1993) J Immunol 150:4550-4555).Receptor-bearing cells are incubated with the labeled signalingmolecules. The cells are then washed to removed unbound molecules, andreceptor-bound labeled molecule is quantified. The data obtained usingdifferent concentrations of HR is used to calculate values for thenumber, affinity, and association of other members of the receptorcomplex.

Labeled HR is also useful as a reagent for purification of theparticular molecule(s) of this complex with which it interacts. In oneembodiment of affinity purification, HR is covalently coupled to achromatography column. Cells and their membranes are extracted, HA isremoved and various HA-free subcomponents are passed over the column.HR-associated molecules bind to the column by virtue of their biologicalaffinity. The HR-complex is recovered from the column, dissociated andthe recovered molecule is subjected to N-terminal protein sequencing.This amino acid sequence is then used to identify the molecule or todesign degenerate oligonucleotide probes for cloning the gene from anappropriate cDNA library.

In an alternate method, mRNA is obtained from HR-complex-bearing cellsand made into a cDNA library. The library is transfected into apopulation of cells, and cells expressing the associated molecule(s) areselected using fluorescently labeled HR. The molecule is identified byrecovering and sequencing the recombinant DNA from the highly labeledcells.

In another alternate method, antibodies are raised against HR,specifically monoclonal antibodies. The monoclonal antibodies arescreened to identify those which inhibit the binding of labeled HR.These monoclonal antibodies are then used in affinity purification orexpression cloning of the associated signaling molecule.

Other soluble binding molecules are identified in a similar manner.Labeled HR is incubated with extracts or other appropriate materialsderived from HUVEC cells. After incubation, HR complexes (which arelarger than the size of purified HR molecule) are identified by a sizingtechnique such as size exclusion chromatography or density gradientcentrifugation and are purified by methods known in the art. The solublebinding protein(s) are subjected to N-terminal sequencing to obtaininformation sufficient for database identification, if the solubleprotein is known, or for cloning, if the soluble protein is unknown.

XVI Use and Administration of HR

Antibodies, inhibitors, or antagonists of HR (or other treatments forexcessive HR production, hereinafter abbreviated TEHR), can providedifferent effects when administered therapeutically. TEHRs will beformulated in a nontoxic, inert, pharmaceutically acceptable aqueouscarrier medium preferably at a pH of about 5 to 8, more preferably 6 to8, although the pH may vary according to the characteristics of theantibody, inhibitor, or antagonist being formulated and the condition tobe treated. Characteristics of TEHR include solubility of the molecule,half-life and antigenicity/immunogenicity; these and othercharacteristics may aid in defining an effective carrier. Native humanproteins are preferred as TEHRs, but organic or synthetic moleculesresulting from drug screens may be equally effective in particularsituations.

TEHRs may be delivered by known routes of administration including butnot limited to topical creams and gels; transmucosal spray and aerosol,transdermal patch and bandage; injectable, intravenous and lavageformulations; and orally administered liquids and pills particularlyformulated to resist stomach acid and enzymes. The particularformulation, exact dosage, and route of administration will bedetermined by the attending physician and will vary according to eachspecific situation.

Such determinations are made by considering multiple variables such asthe condition to be treated, the TEHR to be administered, and thepharmacokinetic profile of the particular TEHR. Additional factors whichmay be taken into account include disease state (e.g. severity) of thepatient, age, weight, gender, diet, time of administration, drugcombination, reaction sensitivities, and tolerance/response to therapy.Long acting TEHR formulations might be administered every 3 to 4 days,every week, or once every two weeks depending on half-life and clearancerate of the particular TEHR.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.It is anticipated that different formulations will be effective fordifferent TEHRs and that administration targeting metastatic cancers maynecessitate delivery in a manner different from that being delivered tovascular endothelial cells.

It is contemplated that conditions or diseases which activate leukocytesmay precipitate damage that is treatable with TEHRs. These conditions ordiseases may be specifically diagnosed by the tests discussed above, andsuch testing should be performed in suspected cases of viral or otherinfections; angiogenesis of cancerous tissues; invasive leukemias andlymphomas; or other physiologic/pathologic problems which deviate fromnormal development and result in metastatic cell migration,proliferation and differentiation.

All publications and patents mentioned in the above specification areherein incorporated by reference. The foregoing written specification isconsidered to be sufficient to enable one skilled in the art to practicethe invention. Indeed, various modifications of the above describedmodes for carrying out the invention which are obvious to those skilledin the field of molecular biology or related fields are intended to bewithin the scope of the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 3                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1056 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: Human Umbilical Vein Endothelial Cell                            (B) CLONE: 39200                                                              (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..1056                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGCAAAACTTAAAACAGAAGTTTATTCTTGAACAACAGGAACGTGAA48                            MetGlnAsnLeuLysGlnLysPheIleLeuGluGlnGlnGluArgGlu                              151015                                                                        AAGCTTCAACAAAAAGAATTACAAATTGATTCACTTCTGCAACAAGAG96                            LysLeuGlnGlnLysGluLeuGlnIleAspSerLeuLeuGlnGlnGlu                              202530                                                                        AAAGAATTATCTTCGAGTCTTCATCAGAAGCTCTGTTCTTTTCAAGAG144                           LysGluLeuSerSerSerLeuHisGlnLysLeuCysSerPheGlnGlu                              354045                                                                        GAAATGGCTAAAGAGAAGAATCTGTTTGAGGAAGAATTAAAGCAAACA192                           GluMetAlaLysGluLysAsnLeuPheGluGluGluLeuLysGlnThr                              505560                                                                        CTGGATGAGCTTGATAAATTACAGCAAAAGGAGGAACAAGCTGAAAGG240                           LeuAspGluLeuAspLysLeuGlnGlnLysGluGluGlnAlaGluArg                              65707580                                                                      CTGGTCAAGCAATTGGAAGAGGAAGCAAAATCTAGAGCTGAAGAATTA288                           LeuValLysGlnLeuGluGluGluAlaLysSerArgAlaGluGluLeu                              859095                                                                        AAACTCCTAGAAGAAAAGCTGAAAGGGAAGGAGGCTGAACTGGAGAAA336                           LysLeuLeuGluGluLysLeuLysGlyLysGluAlaGluLeuGluLys                              100105110                                                                     AGTAGTGCTGCTCATACCCAGGCCACCCTGCTTTTGGAGGAAAAGTAT384                           SerSerAlaAlaHisThrGlnAlaThrLeuLeuLeuGluGluLysTyr                              115120125                                                                     GACAGTATGGTGCAAAGCCTTGAAGATGTTACTGCTCAATTTGAAAGC432                           AspSerMetValGlnSerLeuGluAspValThrAlaGlnPheGluSer                              130135140                                                                     TATAAAGCGTTAACAGCCAGTGAGATAGAAGATCTTAAGCTGGAGAAC480                           TyrLysAlaLeuThrAlaSerGluIleGluAspLeuLysLeuGluAsn                              145150155160                                                                  TCATCATTACAGGAAAAAGTGGCCAAGGCTGGGAAAAATGCAGAGGAT528                           SerSerLeuGlnGluLysValAlaLysAlaGlyLysAsnAlaGluAsp                              165170175                                                                     GTTCAGCATCAGATTTTGGCAACTGAGAGCTCAAATCAAGAATATGTA576                           ValGlnHisGlnIleLeuAlaThrGluSerSerAsnGlnGluTyrVal                              180185190                                                                     AGGATGCTTCTAGATCTGCAGACCAAGTCAGCACTAAAGGAAACAGAA624                           ArgMetLeuLeuAspLeuGlnThrLysSerAlaLeuLysGluThrGlu                              195200205                                                                     ATTAAAGAAATCACAGTTTCTTTTCTTCAAAAAATAACTGATTTGCAG672                           IleLysGluIleThrValSerPheLeuGlnLysIleThrAspLeuGln                              210215220                                                                     AACCAACTCAAGCAACAGGAGGAAGACTTTAGAAAACAGCTGGAAGAT720                           AsnGlnLeuLysGlnGlnGluGluAspPheArgLysGlnLeuGluAsp                              225230235240                                                                  GAAGAAGGAAGAAAAGCTGAAAAAGAAAATACAACAGCAGAATTAACT768                           GluGluGlyArgLysAlaGluLysGluAsnThrThrAlaGluLeuThr                              245250255                                                                     GAAGAAATTAACAAGTGGCGTCTCCTCTATGAAGAACTATATAATAAA816                           GluGluIleAsnLysTrpArgLeuLeuTyrGluGluLeuTyrAsnLys                              260265270                                                                     ACAAAACCTTTTCAGCTACAACTAGATGCTTTTGAAGTAGAAAAACAG864                           ThrLysProPheGlnLeuGlnLeuAspAlaPheGluValGluLysGln                              275280285                                                                     GCATTGTTGAATGAACATGGTGCAGCTCAGGAACAGCTAAATAAAATA912                           AlaLeuLeuAsnGluHisGlyAlaAlaGlnGluGlnLeuAsnLysIle                              290295300                                                                     AGAGATTCATATGCTAAATTATTGGGTCATCAGAATTTGAAACAAAAA960                           ArgAspSerTyrAlaLysLeuLeuGlyHisGlnAsnLeuLysGlnLys                              305310315320                                                                  ATCAAGCATGTTGTGAAGTTGAAAGATGAAAATAGCCAACTCAAATCG1008                          IleLysHisValValLysLeuLysAspGluAsnSerGlnLeuLysSer                              325330335                                                                     GAAGTATCAAAACTCCGCTGTCAGCTTGCTAAAAAAAAAACAAAGTGA1056                          GluValSerLysLeuArgCysGlnLeuAlaLysLysLysThrLys*                                340345350                                                                     (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 351 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetGlnAsnLeuLysGlnLysPheIleLeuGluGlnGlnGluArgGlu                              151015                                                                        LysLeuGlnGlnLysGluLeuGlnIleAspSerLeuLeuGlnGlnGlu                              202530                                                                        LysGluLeuSerSerSerLeuHisGlnLysLeuCysSerPheGlnGlu                              354045                                                                        GluMetAlaLysGluLysAsnLeuPheGluGluGluLeuLysGlnThr                              505560                                                                        LeuAspGluLeuAspLysLeuGlnGlnLysGluGluGlnAlaGluArg                              65707580                                                                      LeuValLysGlnLeuGluGluGluAlaLysSerArgAlaGluGluLeu                              859095                                                                        LysLeuLeuGluGluLysLeuLysGlyLysGluAlaGluLeuGluLys                              100105110                                                                     SerSerAlaAlaHisThrGlnAlaThrLeuLeuLeuGluGluLysTyr                              115120125                                                                     AspSerMetValGlnSerLeuGluAspValThrAlaGlnPheGluSer                              130135140                                                                     TyrLysAlaLeuThrAlaSerGluIleGluAspLeuLysLeuGluAsn                              145150155160                                                                  SerSerLeuGlnGluLysValAlaLysAlaGlyLysAsnAlaGluAsp                              165170175                                                                     ValGlnHisGlnIleLeuAlaThrGluSerSerAsnGlnGluTyrVal                              180185190                                                                     ArgMetLeuLeuAspLeuGlnThrLysSerAlaLeuLysGluThrGlu                              195200205                                                                     IleLysGluIleThrValSerPheLeuGlnLysIleThrAspLeuGln                              210215220                                                                     AsnGlnLeuLysGlnGlnGluGluAspPheArgLysGlnLeuGluAsp                              225230235240                                                                  GluGluGlyArgLysAlaGluLysGluAsnThrThrAlaGluLeuThr                              245250255                                                                     GluGluIleAsnLysTrpArgLeuLeuTyrGluGluLeuTyrAsnLys                              260265270                                                                     ThrLysProPheGlnLeuGlnLeuAspAlaPheGluValGluLysGln                              275280285                                                                     AlaLeuLeuAsnGluHisGlyAlaAlaGlnGluGlnLeuAsnLysIle                              290295300                                                                     ArgAspSerTyrAlaLysLeuLeuGlyHisGlnAsnLeuLysGlnLys                              305310315320                                                                  IleLysHisValValLysLeuLysAspGluAsnSerGlnLeuLysSer                              325330335                                                                     GluValSerLysLeuArgCysGlnLeuAlaLysLysLysThrLys                                 340345350                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 477 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: mouse                                                            (B) CLONE: GI 53979                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetGlnIleLeuThrGluArgLeuAlaLeuGluArgGlnGluTyrGlu                              151015                                                                        LysLeuGlnGlnLysGluLeuGlnSerGlnSerLeuLeuGlnGlnGlu                              202530                                                                        LysGluLeuSerAlaArgLeuGlnGlnGlnLeuCysSerPheGlnGlu                              354045                                                                        GluMetThrSerGluLysAsnValPheLysGluGluLeuLysLeuAla                              505560                                                                        LeuAlaGluLeuAspAlaValGlnGlnLysGluGluGlnSerGluArg                              65707580                                                                      LeuValLysGlnLeuGluGluGluArgLysSerThrAlaGluGlnLeu                              859095                                                                        ThrArgLeuAspAsnLeuLeuArgGluLysGluValGluLeuGluLys                              100105110                                                                     HisIleAlaAlaHisAlaGlnAlaIleLeuIleAlaGlnGluLysTyr                              115120125                                                                     AsnAspThrAlaGlnSerLeuArgAspValThrAlaGlnLeuGluSer                              130135140                                                                     ValGlnGluLysTyrAsnAspThrAlaGlnSerLeuArgAspValThr                              145150155160                                                                  AlaGlnLeuGluSerGluGlnGluLysTyrAsnAspThrAlaGlnSer                              165170175                                                                     LeuArgAspValThrAlaGlnLeuGluSerGluGlnGluLysTyrAsn                              180185190                                                                     AspThrAlaGlnSerLeuArgAspValThrAlaGlnLeuGluSerVal                              195200205                                                                     GlnGluLysTyrAsnAspThrAlaGlnSerLeuArgAspValSerAla                              210215220                                                                     GlnLeuGluSerTyrLysSerSerThrLeuLysGluIleGluAspLeu                              225230235240                                                                  LysLeuGluAsnLeuThrLeuGlnGluLysValAlaMetAlaGluLys                              245250255                                                                     SerValGluAspValGlnGlnGlnIleLeuThrAlaGluSerThrAsn                              260265270                                                                     GlnGluTyrAlaArgMetValGlnAspLeuGlnAsnArgSerThrLeu                              275280285                                                                     LysGluGluGluIleLysGluIleThrSerSerPheLeuGluLysIle                              290295300                                                                     ThrAspLeuLysAsnGlnLeuArgGlnGlnAspGluAspPheArgLys                              305310315320                                                                  GlnLeuGluGluLysGlyLysArgThrAlaGluLysGluAsnValMet                              325330335                                                                     ThrGluLeuThrMetGluIleAsnLysTrpArgLeuLeuTyrGluGlu                              340345350                                                                     LeuTyrGluLysThrLysProPheGlnGlnGlnLeuAspAlaPheGlu                              355360365                                                                     AlaGluLysGlnAlaLeuLeuAsnGluHisGlyAlaThrGlnGluGln                              370375380                                                                     LeuAsnLysIleArgAspSerTyrAlaGlnLeuLeuGlyHisGlnAsn                              385390395400                                                                  LeuLysGlnLysIleLysHisValValLysLeuLysAspGluAsnSer                              405410415                                                                     GlnLeuLysSerGluValSerLysLeuArgSerGlnLeuValLysArg                              420425430                                                                     LysGlnAsnGluLeuArgLeuGlnGlyGluLeuAspLysAlaLeuGly                              435440445                                                                     IleArgHisPheAspProSerLysAlaPheCysHisAlaSerLysGlu                              450455460                                                                     AsnPheThrProLeuLysGluGlyAsnProAsnCysCys                                       465470475                                                                     __________________________________________________________________________

We claim:
 1. A purified HR polypeptide whose amino acid sequence isshown in SEQUENCE ID NO 2.